Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
1. The Field of the Invention
This invention relates to the medical arts. In particular, it relates to a method of inhibiting bacterial growth in the gastrointestinal tract of a human or non-human vertebrate by using an antimicrobial agent.
2. Discussion of the Related Art
Antimicrobial or antibiotic agents are used in the treatment of bacterial infections, especially of the gastrointestinal tract. Gastrointestinal infections affect millions of people world-wide, especially children, and pose an increasing health hazard in hospital settings.
Of course, bacteria inhabit healthy intestines to the benefit of their human and animal hosts. Anaerobic bacteria, including the Bacteroides fragilis group and Clostridium species are common members of the intestinal microflora of healthy individuals, and non-toxigenic strains can be transmitted without causing disease symptoms. (B. A. Cunha, Nosocomial diarrhea, Crit. Care Clin. 14(2):329-38 [1998]; H. Kato et al., Application of typing by pulsed-yield gel electrophoresis to the study of Clostridium difficile in a neonatal intensive care unit, J. Clin. Microbiol. 32(9):2067-70 [1997]; V. O. Rotimi and B. I. Duerden, Bacteroides species in the normal neonatal faecal flora, J. Hyg. [Lond.] 87(2):299-304 [1981]; B. I. Duerden, The isolation and identification of Bacteroides spp. From the normal human faecalflora, J. Med. Microbiol. 13(1):69-78 [1980]; S. S. Long and R. M. Swenson, Development of anaerobic flora in healthy newborn infants, J. Pediatr. 91(2):298-301 [1977]). Other pathogenic bacterial strains have an adverse effect on their hosts.
For example, enterotoxigenic strains of Bacteroides fragilis are associated with diarrhea in humans. (S. K. Niyogi et al., Association of enterotoxigenic Bacteroides fragilis with childhood diarrhoea, Indian J. Med. Res. 105:167-69 [1997]; R. B. Sack et al., Enteroloxigenic Bacteroides fragilis: epidemiologic studies of its role as a human diarrhoeal pathogen, J. Diarrhoeal Dis. Res. 10(1):4-9 [1992]). And patients with Crohn's disease were reported to have higher numbers of B. fragilis group bacteria in their intestines than healthy controls. (J. G. Ruseler-van Embden and H.C. Both-patoir, Anaerobic gram-negative faecal flora in patients with Crohn's disease and healthy subjects, Antonie van Leeuwenhoek 49(2):125-32 [1983]).
More prominent agents of gastrointestinal disease than Bacteroides, are the Clostridium species, especially C. difficile and C. perfringens. Clostridium species are gram-positive, spore-forming anaerobes; some strains that colonize the human intestines can, under certain circumstances, release potent protein exotoxins that induce inflammation of the intestinal mucosa. (M. L. Job and N. F. Jacobs, Jr., Drug-induced Clostridium difficile-associated disease, Drug Saf. 17(1):37-46 [1997]). For example, antibiotics and other chemotherapeutic agents can induce the expression of Toxins A and B from Clostridium difficile. (B. A. Cunha [1998]). Agents known to have a high potential to induce C. difficile-associated disease are aminopenicillins, cephalosporins and clindamycin. (M. L. Job and N. F. Jacobs, Jr., Drug-induced Clostridium difficile-associated disease, Drug. Saf. 17(1):37-46 [1997]; Y. Hutin et al., Prevalence of and risk factors for Clostridium difficile colonization at admission to an infectious diseases ward, Clin. Infect. Dis. 24(5):920-24 [1997]; C. D. Settle and M. H. Wilcox [1996]).
In developed countries, the great majority of cases of C. difficile infection are hospital-acquired, and the number of nosocomial clostridial infections is reported to be rising. (C. D. Settle and M. H. Wilcox, Review article: antibiotic-induced Clostridium difficile infection, Aliment. Pharmacol. Ther. 10(6):835-41[1996]; J. S. Brazier, The epidemiology and typing of Clostridium difficile, J. Antimicrob. Chemother. 41 Suppl. C:47-57 [1998]; S. Tabaqchali and M. Wilks, Epidemiological aspects of infections caused by Bacteroides fragilis and Clostridium difficile, Eur. J. Clin. Microbiol. Infect. Dis. 11(11): 1049-57 [1992]; C. R. Clabots et al., Acquisition of Clostridium difficile by hospitalized patients: evidence for colonized new admissions as a source of infection, J. Infect. Dis. 166(3):561-67 [1992]).
A nosocomial pleural infection with C. difficile, following surgical insertion of a chest drain has also been reported (A. J. Simpson et al., Nosocomial empyema caused by Clostridium difficile, J. Clin. Pathol. 49(2):172-73 [1996]), but intestinal infections are the greatest problem.
Nosocomial diarrhea due to gastrointestinal infection with C. difficile has become a major health care problem, causing 20-30% of all nosocomial diarrheas and affecting up to 8% of hospitalized patients. (L. R. Peterson and P. J. Kelly, The role of the clinical microbiology laboratory in the management of Clostridia difficile-associated diarrhea, Infect. Dis. Clin. North Am. 7(2):277-93 [1993]). Clostridium difficile is considered to be the premier cause of diarrhea among hospitalized patients. (M. Delmee et al., Treatment of Clostridium difficile colitis. Summary of a round table held in Brussels on Mar. 3, 1994, Acta Clin. Belg. 50(2):114-116 [1995]).
An infection of C. difficile can add an average of three weeks to a patient's hospital stay. (C. D. Settle and M. H. Wilcox [1996]). Symptoms may include, diarrhea, self-limited colitis, toxic megacolon or potentially lethal fulminant pseudomembranous colitis. Intestinal infection with C. difficile has also been linked to reactive arthritis. (I. H. Kocar et al., Clostridium infection in patients with reactive arthritis of undetermined etiology, Scand. J. Rheumatol. 27(5):357-62 [1998]; R. K. Cleary, Clostridium difficile-associated diarrhea and colitis: clinical manifestations, diagnosis, and treatment, Dis. Colon. Rectum 41(11):1435-49 [1998]). Bacteraemia and subsequent sepsis is another possible complication of intestinal infection by C. difficile. (P. Naaber et al., Bacterial translocation, intestinal microflora and morphological changes of intestinal mucosa in experimental models of Clostridium difficile infection, J. Med. Microbiol. 47(7):591-98 [1998]; R. J. Feldman et al., Bacteremia due to Clostridium difficile: case report and review of extraintestinal C. Difficile infections, Clin. Infect. Dis. 20(6):1560-62 [1995]). In at least one nosocomial outbreak, 17 patients died from C. difficile infection. (C. D. Settle and M. H. Wilcox [1996]). Clostridium difficile intestinal infections in children, unassociated with antibiotic use or hospital stays, can cause chronic diarrhea and failure to grow. (T. E. Liston, Clostridium difficile toxin associated with chronic diarrhea and failure to gain weight, Clin. Pediatr. (Phila.) 22(6):458-60 [1983]).
In developing countries, C. difficile is also thought to be a causal agent of wide-spread acute cliarrheal disease. (S. K. Niyogi et al., Prevalence of Clostridium difficile in hospitalized patients with acute diarrhoea in Calcutta, J. Diarrhoeal Dis. Res. 9(1):16-19 [1991]; S. Q. Akhtar, Isolation of Clostridium difficile from diarrhoea patients in Bangladesh, J. Trop. Med. Hyg. 90(4):189-92 [1987]).
Enterotoxigenic strains of C. perfringens are linked with a significant number of cases of antibiotic-associated diarrhea, especially among elderly hospitalized patients, children, and infants. (A. Wada et al., Nosocomial diarrhoea in the elderly due to enterotoxigenic Clostridium perfringens, Microbiol. Immunol. 40(10):767-71 [1996]; M. M. Brett et al., Detection of Clostridium perfringens and its enterotoxin in cases of sporadic diarrhoea, J. Clin. Pathol. 45(7):609-11 [1992]; S. C. Samuel et al., An investigation into Clostridium perfringens enterotoxin-associated diarrhoea, J. Hosp. Infect. 18(3):219-30 [1991]; S. P. Boriello et al., Epidemiology of diarrhoea caused by enterotoxigenic Clostridium perfringens, J. Med. Microbiol. 20(3):363-72 [1985]; R. Willliams et al., Diarrhoea due to entertoxigenic Clostridium perfringens: clinical features and management of a cluster of 10 cases, Age Aging 14(5):296-302 [1985]). Clostridium perfringens has been implicated as a possible contributor to sudden infant death syndrome (SIDS) in susceptible infants. (R. R. Meer et al., Human disease associated with Clostridium perfringens enterotoxin, Rev. Environ. Contam. Toxicol. 150:75-94 [1997]).
Clostridium perfringens is well known as a causative agent of non-gastrointestinal gangrene, a special problem for many elderly and diabetic patients with poor blood circulation. But also in more extreme cases of gastrointestinal infection, C. perfringens can cause enteritis necroticans, a gangrene of the bowel resulting in necrosis, sepsis, and hemolysis, in humans and domesticated animals. (L. E. Clarke et al., Enteritis necroticans with midgut necrosis caused by Clostridium perfringens, Arch. Surg. 129(5):557-60 [1994]; D. Bueschel et al., Enterotoxigenic Clostridium perfringens type A necrotic enteritis in a foal, J. Am. Vet. Med. Assoc. 213(9):1305-07 [1998]; E. G. Pearson et al., Hemorrhagic enteritis caused by Clostridium perfringens type C in afoal, J. Am. Vet. Med. 188(11):1309-10 [1986]; F. Al-Sheikhy and R. B. Truscott, The interaction of Clostridium perfringens and its toxins in the production of necrotic enteritis of chickens, Avian Dis. 21(2):256-63 [1977]).
Although rare in developed countries, clostridial enteritis necroticans in humans is more common in some developing countries. (D. A. Watson et al., Pig-bel but no pig: enteritis necroticans acquired in Australia, Med. J. Aust. 155(1):47-50 [1991]). In New Guinea, enteritis necroticans, known locally as pigbel, has been a major cause of illness and death among children. (G. W. Lawrence et al., Impact of active immunisation against enteritis necroticans in Papua New Guinea, Lancet 336(8724): 1165-67 [1990]). Clostridium perfringens type C, the etiologic agent of enteritis necroticans, was also isolated from Bangladeshis with bloody or watery diarrheal illness. (F. P. van Loon et al., Clostridium perfringens type C in bloody and watery diarrhea in Bangladesh, Trop. Geogr. Med. 42(2):123-27 [1990]).
Entertoxigenic strains of C. perfringens have also been linked to nosocomial and non-nosocomial outbreaks of food poisoning, due to heat-resistant spores and a rapid growth rate in warm food. (A. M. Pollack and P. M. Whitty, Outbreak of Clostridium perfringens food poisoning, J. Hosp. Infect. 17(3):179-86 [1991]; M. Van Damme-Jongsten et al., Synthetic DNA probes for detection of enterotoxigenic Clostridium perfringens strains isolated from outbreaks of food poisoning, J. Clin. Microbiol. 28(1):131-33 [1990]).
Spores of Clostridium botulinum germinating in warm food can cause another form of food poisoning called botulism. Growing particularly in non-acidic foods lacking nitrites, and protected from oxygen, the vegetative cells of C. botulinum release an exotoxin that when consumed with the food is activated by trypsin in the stomach, and is absorbed intact by the blood stream. The exotoxin binds to nerve cells, preventing the release of the neurotransmitter acetylcholine. Resulting symptoms of botulism include blurred vision, difficulty in swallowing and speaking, and increasing muscular weakness, and usually nausea and vomiting. Death often results from paralysis of the muscles required for breathing. (R. Y. Stanier et al., The Microbial World, 5 th ed., Prentice Hall, Englewood Cliffs, N.J. pp.626-27 [1986]). Clostridium botulinum sometimes colonizes the intestines of infants and can cause infantile botulism, which can lead to respiratory paralysis and sudden infant death.
Botulism is a problem for the food packaging industry. Spores of C. botulinum may not be killed if canning is done at too low a temperature. High temperature autoclave treatment may be unsuitable for some foods Mayonnaise and other non-acidic foods are particularly prone to foster the growth of C. botulinum. Now with increasing health concerns about the use of nitrite as a food preservative, alternative antimicrobial agents are needed against the growth of C. botulinum and other food poisoning bacterial pathogens.
Antimicrobial agents with selective toxicity for a specific spectrum or range of pathogenic microorganisms are well known in the art. One class of antimicrobial agents is the antibiotics, which are compounds, synthesized and excreted by various microorganisms, that are selectively toxic to other microorganisms, specifically bacteria. In addition, some antibiotics can be artificially modified to produce antimicrobial agents that are more effective and/or more able to overcome antibiotic resistance.
The first line antibiotic treatment for diseases associated with gastrointestinal infections of Clostridium has been a 10-day course of metronidazole or vancomycin, which may be administered orally, intravenously, or rectally. (R. K. Cleary [1998]; C. P. Kelly and J. T. LaMont, Clostridium difficile infection, Annu. Rev. Med. 49:375-90 [1998]; C. M. Reinke and C. R. Messick, Update on Clostridium difficile-induced colitis, Part 2, Am. J. Hosp. Pharm. 51(15):1892-1901 [1994]).
Neither of these antibiotics has been completely satisfactory. While metronidazole (flagyl) is effective against obligate anaerobes (e.g., P. Muir et al., Breath hydrogen excretion by healthy cats after oral administration of oxytetracycline and metronidazole, Vet. Rec. 138:635-39 [1996]; B. Lembcke et al., Different actions of neomycin and metronidazole on breath hydrogen (H) exhalation, Z. Gastroenterol. 18(3):155-60 [1980]), it yields an unpleasant after-taste to many patients, even when delivered intravenously. Other common side effects of metronidazole are neuropathy and gastrointestinal distress. Also, metronidazole is a known reproductive toxicant affecting mammalian sperm cells. (R. E. Linder et al., Endpoints of spermatotoxicity in the rat after short duration exposures to fourteen reproductive toxicants, Reprod. Toxicol. 6(6):491-505 [1992]).
On the other hand, a course of vancomycin is prohibitively expensive (10-50 times more expensive than metronidazole), and there are concerns about the rapid development of vancomycin-resistance among pathogenic Clostridium, Enterococcus, Pediococcus, Citrobacter, Klebsiella, Enterobacter, and Staphylococcus species, because the plasmid-borne vancomycin resistance gene (VanR) is readily transmissible. (ASHP therapeutic position statement on the preferential use of metronidazole for the treatment of Clostridium difficile-associated disease, Am. J. Health Syst. Pharm. 55(13):1407-1 [1998]; S. H. Cohen et al., Isolation of a toxin B-deficient mutant strain of Clostridium difficile in a case of recurrent C. difficile-associated diarrhea, Clin. Infect. Dis. 26(2):1250 [1998]; C. Edlund et al., Effect of vancomycin on intestinal flora of patients who previously received antimicrobial therapy, Clin. Infect. Dis. 25(3):729-32 [1997]; C. A. O'Donovan et al., Enteric eradication of vancomycin-resistant Enterococcus faecium with oral bacitracin, Diagn. Microbiol. Infect. Dis. 18(2):105-09 [1994]; E. Yamaguchi et al., Colonization pattern of vancomvcin-resistant Enterococcus faecium, Am. J. Infect. Control 22(4):202-06 [1994]; C. P. Kelly and J. T. LaMont [1998]).
In addition, relapses of clostridial infections occur in about 5-42% of those treated with metronidazole or vancomycin and are believed to be caused by persistent endogenous clostridial spores, which are antibiotic resistant. (S. H. Cohen et al. [1998]; R. Fekety et al., Recurrent Clostridium difficile diarrhea: characteristics of and risk factors for patients enrolled in a prospective, randomized, double-blinded trial, Clin. Infect. Dis. 24(3):324-33 [1997]; S. Johnson et al., Treatment of asymptomatic Clostridium difficile carriers fecal excretors with vancomycin and metronidazole. A randomized, placebo-controlled trial, Ann. Intern. Med. 117(4):297-302; M. J. Zimmerman et al., Review article: treatment of Clostridium difficile infection, Aliment. Pharmacol. Ther. 11(6):1003-12 [1997]).
Teicoplanin is another antibiotic found to be effective against gram positive anaerobes such as Propionibacterium acnes, Clostridium perfringens, C. difficile, and other Clostridium spp., Peptococcus spp., Peptostreptococcus spp. (H. Hassan et al., In vitro activity of teicoplanin, vancomycin, A16686, clindamycin, erythromycin and fusidic acid against anaerobic bacteria, Singapore Med. J. 31(1):56-58 [1990]; The Swedish CDAD Study Group, Treatment of Clostridium difficile associated diarrhea and colitis with an oral preparation of teicoplanin; a dose finding study, Scand. J. Infect. Dis. 26(3):309-16 [1994]; C. Wenisch et al., Comparison of vancomycin, teicoplanin, metronidazole, and fusidic acid for the treatment of Clostridium difficile-associated diarrhea, Clin. Infect. Dis. 22(5):813-18 [1996]).
However, teicoplanin is not widely available. The peptide antibiotic bacitracin is also reported to be effective in treating C. difficile-induced diarrhea, but it is not widely available in an enteric formulation. (M. N. Dudley et al., Oral bacitracin vs. vancomycin therapyfor Clostridium difficile-induced diarrhea, A randomized double-blind trial, Arch. Intern. Med. 146(6):1101-04 [1986]).
Other adjunct treatments are reportedly effective for refractory C. difficile-related disease, including whole-bowel irrigation and enteric administration of the non-pathogenic yeast Saccharomyces boulardii. (C. A. Liacouras and D. F. A. Piccoli, Whole-bowel irrigation as an adjunct to the treatment of chronic, relapsing Clostridium difficile colitis, J. Clin. Gastroenterol. 22(3):186-89 [1996]; C. M. Surawicz, Clostridium difficile disease: diagnosis and treatment, Gastroenterologist 6(1):60-65 [1998]). But such treatments are uncomfortable or distasteful for many patients and are less suitable than easily administered antibiotics as a first line treatment regimen.
Accordingly, there remains a definite need for a modestly priced antimicrobial agent for treating gastrointestinal infections, without the commonly unpleasant side effects and bacteria resistance associated with metronidazole and vancomycin.
Pharmaceutical preparations of 4-(p)-aminosalicylic acid (i.e., 4-ASA or para-aminosalicylic acid) or 4-(p)-aminosalicylate sodium salt (e.g., Nemasol-Sodium.RTM. or Tubasal.RTM.) have been used systemically in cases of tuberculosis as antimicrobial chemotherapeutic agents against Mycobacterium tuberculosis.
On the other hand, the 5-aminosalicylates are known as anti-inflammatory chemotherapeutic agents and have not been used as antimicrobial agents. These compounds include 5-aminosalicylic acid (i.e., 5-ASA, mesalamine, or mesalazine) and conjugated derivatives thereof, known for their anti-inflammatory properties. These anti-inflammatory agents are commercially available in various pharmaceutical preparations such as Asacol.RTM., Rowasa.RTM., Claversal.RTM., Pentasa.RTM., Salofalk.RTM., Dipentum.RTM., Azulfidine.RTM. (SAZ) and others.
5-Aminosalicylates have been used widely to reduce mucosal inflammation in inflammatory bowel disease, ulcerative colitis and Crohn's disease. (S. B. Hanauer and FB. Baert, Medical Therapy of Inflammatory Bowel Disease, Inflamm. Bowel Dis. 78(6):1413-25 [1994]; C. J. Mulder et al., Drug therapy. dose-response relationship of oral mesalazine in inflammatory bowel disease, Mediators Inflamm. 7(3):135-36 [1998]; W. Kruis et al., Olsalazine versus mesalazine in the treatment of mild to moderate ulcerative colitis, Aliment. Pharmacol.12(8):707-15 [1998]; J. N. Healey, Gastrointestinal transit and release of mesalazine tablets in patients with inflammatory bowel disease, Scand. J. Gastroenterol. 172:47-51 [1990]).
The mechanism underlying the anti-inflammatory properties of the 5-aminosalicylates is unknown, but it may result from their ability to inhibit oxidation at the surface of endothelial membranes, perhaps through radical scavenging, and to prevent lipid peroxidation. (D. C. Pearson et al., The anti-oxidant properties of5-aminosalicylic acid, Free Radic. Biol. Med. 21(3):367-73 [1996]). 5-Aminosalicylates may be able to act synergistically with endogenous antioxidants such as alpha-tocopherol to prevent the oxidative damage implicated in the pathogenesis of inflammatory bowel diseases. (E. Goncalves et al., Antioxidant activity of 5-aminosalicylic acid against peroxidation of phosphotidylcholine liposomes in the presence of alpha-tocopherol: a synergistic effect?, Free Radic. Res. 29(1):53-66 [1998]).
There are several reports that 5-aminosalicylic acid also inhibits fimbriae-mediated cellular adhesion by enteroaggregative Escherischia coil strains, associated with both acute and persistent diarrhea in infants and children. (G. Kang et al., Salicylate inhibits fimbriae mediated Hep-2 cell adherence ofand haemagglutination by enteroaggregative Escherischia coli, FEMS Microbiol. Lett. 166(2):257-65 [1998]; D. Law and H. Chart, Enteroaggregative Escherischia coil, J. Appl. Microbiol. 84(5):685-97 [1998]; Y. Germani et al., Prevalence of enteropathogenic, enteroaggregative Escherischia coli among isolates from children with diarrhea in New Caledonia, J. Infect. Dis. 174(5):124-26 [1996]; S. Knutton et al., Ability of enteroaggregative Escherischia coil strains to adhere in vitro to human intestinal mucosa, Infect. Immun. 60(5):2083-91 [1992]).
Antimicrobial growth inhibitory properties of the 5-aminosalicylates and other features and advantages of the present invention will be described herein.